The placenta is the nexus between the fetus and mother; placental health is absolutely central and critical to both fetal and maternal health. The placenta is the source of nutrition and oxygenation for the fetus, allows the fetus to eliminate waste, protects it from toxins, environmental exposures, infectious agents and rejection by the maternal immune system, and produces hormones that support the pregnancy. Abnormalities in placental structure and function underlie complications associated with pregnancy, including preterm birth, miscarriage and stillbirth, and hypertensive disorders such as preeclampsia. Therefore, monitoring placental function is necessary in order to prevent adverse outcomes for the mother and the fetus. Early detection of changes in placental function provides the opportunity for increased pregnancy surveillance and treatment. As is the case with every other organ, the functional state of the placenta is reflected by changes in the expression of coding and non-coding genes. Thus, monitoring the transcriptome of the placenta allows for a comprehensive assessment of placental function. The challenge is to be able to assess placental transcriptome without using invasive methods (e.g. placental biopsy) because these methods pose a risk to the pregnancy. To be clinically relevant, this method must be robust, reliable and able to use on large numbers of clinical samples at reasonable costs, and be able to monitor the full placental transcriptome. Unfortunately, these goals are not possible with existing technology. Our laboratory has developed an RNA sequencing method to comprehensively and quantitatively profile extracellular RNA (exRNA), specifically, placental microRNAs (miRNAs), found in maternal circulation. In the proposed project, we will advance this method and develop technology that will enable non-invasive monitoring of the placental transcriptome through the maternal blood and urine. In Aim 1, we will develop two new technologies, automated exRNA Isolation (AxRI) and total short RNA sequencing (tsRNAseq) that will enable isolation and sequencing of coding and non-coding exRNAs from the maternal blood and urine. In Aim 2, we will apply these new technologies to monitor placenta-specific exRNAs in the maternal blood and urine of term and first and second trimester pregnancies; these earlier time points are important because these is the gestational ages when changes in the transcriptome will be the most clinically relevant and biologically informative. In Aim 3, we will define the precise cell-origin of placenta-specific exRNAs to provide contextual information to analysis of the exRNAs. Therefore, the scope of this project provides the unprecedented opportunity to leverage cutting-edge technologies for exRNA isolation and detection, sequencing, and bioinformatics, in order to develop a tool to non-invasively monitor the transcriptome of the placenta in real time. This tool can then be used to gain insights into pathogenesis of pregnancy-related diseases, identify high-risk pregnancies before the onset of end-organ damage and, ultimately, provide therapeutic interventions to improve maternal and neonatal health.